Flow cytometric detection of activated mouse integrin αIIbβ3 with a novel monoclonal antibody
W Bergmeier, V Schulte, G Brockhoff… - … : The Journal of the …, 2002 - Wiley Online Library
W Bergmeier, V Schulte, G Brockhoff, U Bier, H Zirngibl, B Nieswandt
Cytometry: The Journal of the International Society for Analytical …, 2002•Wiley Online LibraryAbstract Background Integrin αIIbβ3 mediates platelet adhesion and aggregation and plays
a crucial role in thrombosis and hemostasis. αIIbβ3 is expressed in a low affinity state on
resting platelets. Upon platelet activation, αIIbβ3 shifts to a high affinity conformation that
efficiently binds its ligands. On human platelets, the high affinity conformation of αIIbβ3 is
detected by the monoclonal antibody (mAb), PAC‐1. However, a reagent with binding
specificity to high affinity mouse αIIbβ3 has not been described so far. Methods A novel rat …
a crucial role in thrombosis and hemostasis. αIIbβ3 is expressed in a low affinity state on
resting platelets. Upon platelet activation, αIIbβ3 shifts to a high affinity conformation that
efficiently binds its ligands. On human platelets, the high affinity conformation of αIIbβ3 is
detected by the monoclonal antibody (mAb), PAC‐1. However, a reagent with binding
specificity to high affinity mouse αIIbβ3 has not been described so far. Methods A novel rat …
Background
Integrin αIIbβ3 mediates platelet adhesion and aggregation and plays a crucial role in thrombosis and hemostasis. αIIbβ3 is expressed in a low affinity state on resting platelets. Upon platelet activation, αIIbβ3 shifts to a high affinity conformation that efficiently binds its ligands. On human platelets, the high affinity conformation of αIIbβ3 is detected by the monoclonal antibody (mAb), PAC‐1. However, a reagent with binding specificity to high affinity mouse αIIbβ3 has not been described so far.
Methods
A novel rat mAb directed against mouse αIIbβ3 (JON/A) was generated and characterized. JON/A was conjugated with fluorescein isothiocyanate (JON/AFITC) or with R‐phycoerythrin (JON/APE) and used for flow cytometric analysis of mouse platelets.
Results
Although JON/AFITC bound to resting and activated platelets, virtually no binding of the larger JON/APE to resting platelets was detectable. However, strong binding of JON/APE occurred on platelet activation in a dose‐dependent manner. Binding of JON/APE required extracellular free calcium and was irreversible, thereby stabilizing the high affinity conformation of αIIbβ3.
Conclusion
JON/APE is the first tool for direct assessment of integrin αIIbβ3 activation in mice. Furthermore, JON/AFITC and JON/APE provide the first examples of fluorescent antibody derivatives with identical antigenic specificitiy that allow the discrimination between the resting and the activated state of an integrin. Cytometry 48:80–86, 2002. © 2002 Wiley‐Liss, Inc.
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